Optogenetic control of the lac operon for bacterial chemical and protein production

Abstract

Control of the lac operon with isopropyl β-d-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.

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Fig. 1: OptoLAC circuits design.
Fig. 2: OptoLAC circuit characterization.
Fig. 3: Dynamic control of isobutanol production using OptoLAC circuits.
Fig. 4: Dynamic control of mevalonate production using OptoLAC circuits and scale-up.
Fig. 5: Light-controlled recombinant protein production in E. coli B strain (OptoBL).

Data availability

All data supporting the findings of this study are available within the paper and Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank M. Brynildsen for strain MG1655 ΔlacI::FRT-KanR-FRT, A. Möglich for plasmids pDusk and pDawn, J. Keasling for plasmid pMevT and J. Liao for plasmids pSA65 and pSA69. We are very grateful to W. Mok and M. Brynildsen for advice and troubleshooting regarding E. coli protocols. We thank C. DeCoste, K. Rittenbach and the Princeton Molecular Biology Flow Cytometry Resource Center for assistance with flow cytometry experiments. J.L.A. is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research award no. DE-SC0019363, the NSF CAREER Award CBET-1751840, The Pew Charitable Trusts, The Eric and Wendy Schmidt Transformative Technology Fund Award and the Camille Dreyfus Teacher-Scholar Award.

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Authors

Contributions

M.A.L. and J.L.A. conceived this project and designed the experiments. M.A.L., S.S.I. and C.C.-L. constructed the strains and plasmids. M.A.L. and S.S.I. performed the experiments shown in Figs. 1 and 2. M.A.L., E.M.Z. and H.K. performed the experiments shown in Figs. 3 and 4. M.A.L., C.C.-L. and C.D. performed the experiments shown in Fig. 5. M.A.L. performed experiments shown in Extended Data Figs. 110. C.C.-L. and C.D. performed experiments shown in Extended Data Figs. 2 and 9. M.A.L., C.C.-L. and J.L.A. analyzed the data and wrote the manuscript. J.L.A. supervised and funded the project.

Corresponding author

Correspondence to José L. Avalos.

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Competing interests

J.L.A., M.A.L. and C.C.-L. have filed a patent application (‘Optogenetic circuits for controlling chemical and protein production in Escherichia coli’, US patent application 62,935,267) describing the OptoLAC circuit design and application for chemical and recombinant protein production.

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Extended data

Extended Data Fig. 1 Development of OptoLAC circuits.

a, GFP expression in blue light or darkness from the constitutive PBBA promoter (EMAL231) or from PT5-lacO in a strain in which lacI expression is controlled by pDawn (EMAL57). P = 0.07118. b, GFP expression in blue light or darkness from PBBA (EMAL231) or from PT5-lacO in strains containing the pDawn system controlling LacI fused to SsrA tags terminating in LAA (EMAL68, which gave rise to OptoLAC1), AAV (EMAL69, which gave rise to OptoLAC2), or ASV (EMAL71). From left to right: P < 0.00001, P < 0.00001, P = 0.00536. c, GFP expression in blue light or darkness driven by the PFixK2 (pMAL441) or PFixK2-lacO (pMAL442) promoter using pDusk (EMAL152 and EMAL153, respectively). P = 0.000037. **P < 0.01, ***P < 0.001. Statistics are derived using a two-sided t-test. All data shown as median values of 10,000 single-cell flow cytometry events; error bars represent one standard deviation of n = 3 biologically independent samples; open circles represent individual data points. Data are representative of n = 3 independent experiments. Source data

Extended Data Fig. 2 Quantification of LacI protein levels for OptoLAC circuits.

Integrated peak volumes of LacI protein levels quantified via Western blot. From left to right: P = 0.00102, P = 0.000041. Loading controls and uncropped blots used for quantification (samples derived from the same experiment and processed in parallel) are provided as source data. ** P < 0.01, ***P < 0.001. Statistics are derived using a two-sided t-test. All data shown as mean values; error bars represent the standard deviation of n = 3 biologically independent samples; open circles represent individual data points. Data are representative of n = 2 independent experiments. Source data

Extended Data Fig. 3 Response of OptoLAC circuits to IPTG.

GFP expression from PT5-lacO controlled by OptoLAC1 (orange, EMAL68), OptoLAC2 (purple, EMAL69), or OptoLAC3 (gray, EMAL230) under blue light with different concentrations of IPTG added at the time of inoculation. From left to right: P < 0.00001, P = 0.000076, P = 0.00692, P = 0.000025. **P < 0.01, ***P < 0.001. Statistics are derived using a two-sided t-test. All data shown as median values of 10,000 single-cell flow cytometry events; error bars represent one standard deviation of n = 3 biologically independent samples; open circles represent individual data points. Data are representative of n = 3 independent experiments. Source data

Extended Data Fig. 4 Response of OptoLAC circuits to low temperatures.

GFP expression at 18 °C from PBBA (EMAL231) or PT5-lacO controlled by OptoLAC1 (EMAL68), OptoLAC2 (EMAL69), or OptoLAC3 (EMAL230) under blue light or darkness. From left to right: P < 0.00001, P = 0.000014, P = 0.0222. *P < 0.05, ***P < 0.001. Statistics are derived using a two-sided t-test. All data shown as median values of 10,000 single-cell flow cytometry events; error bars represent one standard deviation of n = 3 biologically independent samples; open circles represent individual data points. Data are representative of n = 3 independent experiments. Source data

Extended Data Fig. 5 Tunability of OptoLAC circuits with light intensity.

GFP expression from the constitutive PBBA promoter (EMAL231) or from PT5-lacO controlled by OptoLAC1 (EMAL68) or OptoLAC3 (EMAL230) under continuous blue light of differing intensities or darkness. From left to right: P = 0.0189, P = 0.00314, P < 0.00001, P < 0.00001. *P < 0.05, **P < 0.01, ***P < 0.001. Statistics are derived using a two-sided t-test. All data shown as median values of 10,000 single-cell flow cytometry events; error bars represent one standard deviation of n = 3 biologically independent samples; open circles represent individual data points. Data are representative of n = 3 independent experiments. Source data

Extended Data Fig. 6 Spatial control of GFP expression on an LB agar plate.

LB agar plate containing a lawn of EMAL68 (OptoLAC1 driving GFP) illuminated with a projection of a tiger image. Scale bar: 1 cm. Data are representative of n = 2 independent experiments.

Extended Data Fig. 7 Growth curves of K strains containing OptoLAC circuits under different induction conditions.

ac, OD600 measurements for strains containing OptoLAC1 (EMAL68; orange circle), OptoLAC2 (EMAL69; purple triangle), OptoLAC3 (EMAL230; gray square), or an IPTG-induced control (EMAL77; green diamond) using different induction conditions, shown on top of each graph as uninduced (blue) or induced (gray). a, Cultures of each strain grown under continuous blue light for 4 hours before switching them to darkness (OptoLAC circuits) or adding 1 mM IPTG (IPTG control). From left to right: P = 0.00827, P = 0.1472. b, Cultures grown entirely uninduced under continuous blue light (OptoLAC circuits) or without IPTG (IPTG control). From left to right: P = 0.00275, P = 0.0508. c, Cultures grown constitutively induced in darkness (OptoLAC circuits) or with 1 mM IPTG (IPTG control). From left to right: P = 0.000393, P = 0.00145, P = 0.33. **P < 0.01, ***P < 0.001. Statistics are derived using a two-sided t-test. All data shown as mean values; error bars represent the standard deviation of n = 3 biologically independent samples; open circles represent individual data points. Data are representative of n = 2 independent experiments. Source data

Extended Data Fig. 8 Optimization of the cell density of induction with IPTG for chemical production.

a, Isobutanol production from pMAL534 by EMAL201 when induced with 1 mM IPTG at different cell densities. P = 0.819. b, Mevalonate production from pMAL487 by EMAL135 when induced with 1 mM IPTG at different cell densities. P = 0.02185. *P < 0.01. Statistics are derived using a two-sided t-test. All data shown as mean values; error bars represent the standard deviation of n = 4 biologically independent samples; open circles represent individual data points. Data are representative of n = 2 independent experiments. Source data

Extended Data Fig. 9 Optimization of YFP and FdeR production using OptoLAC circuits.

a, YFP production when inducing at different cell densities (ρs) by switching cultures from blue light to darkness using OptoLAC1B (EMAL284, top panel) or adding IPTG (EMAL283, bottom panel). NI = Not induced: kept under blue light or no IPTG added. b, Comparison of FdeR production between OptoLAC1B (EMAL335) and OptoLAC2B (EMAL336) cultured under continuous blue light for 12 hours. c, FdeR production when inducing at different cell densities (ρs) by adding IPTG (EMAL329, top panel) or switching cultures from blue light to darkness using OptoLAC2B (EMAL336, bottom panel). NI = Not induced: kept under blue light or no IPTG added. All samples were resolved via SDS-PAGE (12% polyacrylamide). d, Tunability of YFP production using different doses of light or concentrations of IPTG, resolved and quantified via Western blot. From left to right: P = 0.0201, P = 0.000383, P = 0.000206. Loading controls and uncropped gels and blots, including those used for quantification in d (samples derived from the same experiment and processed in parallel) are provided as source data. *P < 0.05, ***P < 0.001. Statistics are derived using a two-sided t-test. All data shown as mean values; error bars represent the standard deviation of three biologically independent samples; open circles represent individual data points. Data are representative of n = 2 independent experiments. Source data

Extended Data Fig. 10 Growth curves of B strains containing OptoLAC circuits under different induction conditions.

a, b, Time course of OD600 readings for BL21 DE3 (EMAL283; orange square), Rosetta 2 (EMAL276; green diamond), and OptoBL containing OptoLAC1B (EMAL284; black circle without IPTG, yellow circle with IPTG), containing plasmids for YFP production, grown under different induction conditions, which are shown on top of each graph as uninduced (blue) or induced (gray). a, Cultures from each strain grown under blue light before switching to the dark (OptoLAC1B) or adding IPTG. From left to right: P < 0.0122, P = 0.0946. b, Cultures grown entirely uninduced under blue light (OptoLAC1B), without IPTG, or OptoLAC1B with blue light and IPTG. From left to right: P = 0.00342, P = 0.0017. *P < 0.05, **P < 0.01. Statistics are derived using a two-sided t-test. All data shown as mean values; error bars represent the standard deviation of n = 3 biologically independent samples; open circles represent individual data points. Data are representative of n = 2 independent experiments. Source data

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Lalwani, M.A., Ip, S.S., Carrasco-López, C. et al. Optogenetic control of the lac operon for bacterial chemical and protein production. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-020-0639-1

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